Invasive fungal infections (e.g. Candida spp., Aspergillus spp., Fusarium spp., Zygomycetes spp.) (Walsh, 1992) have emerged during the past two decades as important pathogens causing formidable morbidity and mortality in an increasingly diverse and progressively expanding population of immunocompromised patients. Those with the acquired immune deficiency syndrome (AIDS) constitute the most rapidly growing group of patients at risk for life-threatening mycosis. But fungal Infections have also increased in frequency in several populations of other susceptible hosts, including very-low-birth-weight infants, cancer patients receiving chemotherapy, organ transplant recipients, burn patients and surgical patients with complications.
These fungal infections are not limited to humans and other mammals, but are also important in plants where they can cause diseases or cause the production of unwanted compounds (e.g. Fusarium spp., Aspergillus spp., Botritis spp., Cladosporium spp.).
Although recent advances in antifungal chemotherapy have had an impact on these mycoses, expanding populations of immunocompromised patients will require newer approaches to antifungal therapy. The discovery of novel antifungal agents is thus an essential element of any new antifungal therapy.
Classical approaches for identifying antifungal compounds have relied almost exclusively on inhibition of fungal or yeast growth as an endpoint. Libraries of natural products, semi-synthetic, or synthetic chemicals are screened for their ability to kill or arrest growth of the target pathogen or a related nonpathogenic model organism. These tests are cumbersome and provide no information about a compound's mechanism of action. The promising lead compounds that emerge from such screens must then be tested for possible host-toxicity and detailed mechanism of action studies must subsequently be conducted to identify the affected molecular target.
Cells from multicellular organisms can commit suicide in response to specific signals or injury by an intrinsic program of cell death. Apoptosis is a form of programmed cell death which leads to elimination of unnecessary or damaged cells. Cells that are either unwanted or potentially harmful to the organism undergo the apoptotic process and show events like cell shrinkage, chromatin condensation, cytoplasmic condensation, digestion of nuclear DNA, loss of mitochondrial membrane potential, plasma membrane blebbing and phagocytosis of the cell debris (Schwartz, et al. 1993). The Bcl-2 family of proteins is centrally involved in the control of the programmed cell death process (PCD). Proteins of this group belong either to the inhibitors of cell death (Bcl-2, Bcl-XL) or to the group of proteins promoting apoptosis (Bax, Bak) (Oltvai and Korsmeyer 1994; Knudson and Korsmeyer 1997; Reed et al. 1998). The ability of the Bcl-2 family of proteins to regulate life and death of a cell is conserved across evolution. Finding of homologues of PCD regulatory genes in plants and animals suggests the possibility that some functions involved in this process may originally have evolved in unicellular organisms, before a divergent development between the plant and the animal kingdom had happened (Apte et al. 1995).
Expression of the pro-apoptotic human or mouse Bax protein in Saccharomyces cerevisiae did induce cell death in this budding yeast (Sato et al. 1994; Greenhalf et al. 1996; Zha et al. 1996). It was initially described as a process that resembled autophagy with dissolution of the internal organelles and vacuolisation. The apoptotic features characteristic for multicellular eucaryotic cells like morphological changes In nuclear shape and chromatin condensation, were not observed in this yeast (Zha et al. 1996). It was therefore suggested that Bax-induced cell death in S. cerevisiae is due to the toxicity of the Bax protein itself, mediated by a hypothetical pore-formation without any involvement of a death program (Muchmore et al. 1996).
Bax expression in the fission yeast Schizosaccharomyces pombe did in contrast show some of the typical apoptotic changes like DNA fragmentation, chromatin condensation, dissolution of the nuclear envelope and cytosolic vacuolisation, suggesting the presence of the evolutionary conserved PCD pathway in this unicellular eucaryote (Ink et al. 1997; Jurgensmeier et al. 1997). Since it is very unlikely that species dependent differences in the toxicity of the Bax protein are the reason for this observed difference between the two yeasts, a bona fide cell death pathway may well be present in S. cerevisiae. 
Recent findings of a yeast mutant in the cell division cycle gene CDC48 show a number of morphological and molecular features that are considered typical indicators of apoptosis markers in metazoan cells: exposure of phosphatidylserine on the outer leaflet of the cytoplasmic membrane, DNA breakage as well as chromatin condensation and fragmentation, supporting the existence of a basic PCD machinery in this unicellular yeast. This theory was supported by the analysis of a wild type yeast cell expressing the human Bax protein. Comprehensive tests for morphological markers of apoptosis did show a series of changes, identical to morphological markers defining apoptosis (Ligr, Madeo et al. 1998). Recent findings from the same group (Madeo et al., 1999) implicate oxygen stress as a general regulator of apoptosis in yeast but the actual mechanism of Bax lethality in S. cerevisiae remains unclear. It is an aim of the present invention to provide new bax sequences for expression in yeast and fungi and tools for identifying yeast and candida functions in the pathways leading to programmed cell death.
It is an aim of the present invention to provide nucleic acids as well as polypeptides which represent potential molecular targets for the identification of new compounds which can be used in alleviating diseases or conditions associated with yeast or fungal infections.
It is a further aim of the present invention to provide uses of these nucleic acid and polypeptide molecules for treating diseases associated with yeast or fungi or for the preparation of (a) medicament(s) for treating said diseases.
It is also an aim of the invention to provide pharmaceutical compositions and vaccines comprising these nucleic acids or polypeptides.
It is also an aim of the present invention to provide vectors comprising these nucleic acids, as well as host cells transfected or transformed with said vectors.
It is also an aim of the invention to provide antibodies against these polypeptides, which can be used as such, or in a composition as a medicament for treating diseases associated with yeast and fungi.
It is another aim of the invention to provide methods to selectively identify compounds or polypeptides capable of inhibiting or activating expression of the polypeptides of the invention or capable of selectively modulating expression or functionality of such polypeptides. The nucleic acid and polypeptide molecules alternatively can be incorporated into an assay or kit to identify these compounds or polypeptides.
It is also an aim of the invention to provide methods for preventing infection with yeast or fungi.
It is a further aim of the invention to provide human homologues for the nucleic acids and polypeptides of the invention for use in treating proliferative disorders, such as cancer, or for the prevention of apoptosis in certain diseases, or for the preparation of a medicament for treating such disorders or diseases.
All the aims of the present invention have been met by the embodiments as set out below.